DKK1 Inhibits Intracellular Lipid Accumulation In Macrophages And The Underlying Mechanism

BackgroundThe incidence of atherosclerosis increases day by day, with plaque rupture and thrombosis, which induced acute coronary syndrome, myocardial infarction, stroke and other cardiovascular diseases. The pathogenesis of atherosclerosis includes many hypotheses, such as the formation of thrombosis, inflammation, lipid infiltration and reaction to damage theory. Traditionally, we consider atherosclerosis began with the injury of vascular endothelial, then monocyte recruited and migrated into intima to become macrophages, which can recognize lipid and swallow with endocytosis to transform into foam cells. At the same time, white blood cells and smooth muscle cells migrated into the plaque with lipid accumulation to form the necrotic core, resulting in the rupture of the plaque.Vulnerable plaque with a thin fibrous cap, large lipid-rich necrotic core, increased plaque inflammation, positive vascular remodeling, increased vasa-vasorum neovascularization and intra-plaque hemorrhage, is a murder of cardiovascular events. Oxidized low density lipoprotein (ox-LDL) plays an important factor in the development of atherosclerosis. Ox-LDL can damage vascular endothelial, raise monocytes, promote smooth muscle cells proliferation and migration, accelerate the formation of foam cells, increase inflammation reaction, worsen lipid deposition and other harmful enents.It is reported that Wnt signal pathway involves in cell adhesion, stem cell renewal, tumorigenesis and tumor cell differentiation, proliferation, migration and other molecular biological changes. Environmental factors influence the activity of Wnt signal pathway. The ligand of Wnt signal pathway combined with membrane receptors such as LRP5/6, influence the expression of β-catenin, then regulate the target genes of β-catenin, leading the change of function.There are many extracellular antagonists of Wnt signal pathway. DKK family (DKK1-4), a secreted antagonist, involves in the regulation of Wnt signal pathway except DKK3. Mostly, researches focused on the function of DKK1. There is a close relation between DKK1 and cardiovascular diseases. While how ox-LDL influences the expression of DKK 1 in macrophages was unknown.ObjectivesWe investigated whether ox-LDL regulates the expression of DKK1 in macrophages and the roles of LXRs and β-catenin in the regulation induced by ox-LDL.Materials and MethodsMaterialsHuman DKK1 ELISA kit was from R&D Systems (Minneapolis, MN, USA). Antibody for ABCG1 and protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for DKK1, LXRa and LXRβ were from Abcam (Cambridge, UK). Geranylgeranyl pyrophosphate (GGPP), 22-(S)-hydroxycholesterol and 22-(R)-hydroxycholesterol were from Sigma (St. Louis, MO, USA). T0901317 and GW3965 were from Selleckchem (Shanghai).Cell CultureTHP-1 cells obtained from the American Type Culture Collection (ATCC) were grown in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. For THP-1 cell differentiation into macrophages, cells were seeded in culture plates at 2×106 cells/1 ml per well and allowed to adhere and differentiate overnight in the presence of 160 nM phorbolmyristate acetate (PMA).RNA interferenceMacrophages cultured with antibiotic-free medium were transfected with specific siRNA or negative control siRNA (GenePharma, shanghai) by using Lipofectamine 2000. After 6 hr, complete culture medium was replaced. Gene silencing efficiency was determined by western blot analysis.Western blotCells were lysed with RIPA lysis buffer including 1 mM PMSF. Equal amounts of extracted proteins were separated on 10% SDS-PAGE gel and transferred to PVDF membranes for incubation with primary antibodies overnight at 4℃. The bands were visualized by enhanced chemiluminescence reagents and recorded by use of the LAS-4000 luminescent image analyzer (Fujifilm, Stamford, CT, USA).Real-time PCRThe total RNA was extracted from macrophages by use of TranZol reagent. cDNA was prepared with PrimeScript RT reagent kit. Real-time PCR involved use of the SYRB Premix Ex Taq kit (Takara Bio Inc.). The primer sequences were for DKK1, forward,5’-GGGAATTACTGCAAAAATGGAATA-3’, reverse, 5’-ATGACCGGAGACAAACCAGAAC-3’; and β-actin, forward, 5’-CGTGCGTGACATTAAGGAGA-3’, reverse, 5’-CACCTTCACCGTTCCAGTTT-3’,The 2-△△Ct method was used to assess the relative mRNA expression level normalized to that of β-actin.Enzyme-linked immunosobent assay (ELISA)THP-1 differentiated macrophages were stimulated with or without ox-LDL, and medium was collected. DKK1 in the supernatant was measured by ELISA assay according to the manufacturer’s instruction.ImmunoprecipitationDifferentiated THP-1 cells were incubated with or without ox-LDL, then lysed with lysis buffer for western blot analysis and immunoprecipitation. Cell lysates were incubated with cognate antibodies and 50% protein A/G agarose overnight at 4℃ with gentle rotation. Agarose-bound immunoprecipitates were rinsed and underwent SDS-PAGE (2×) before western blot analysis.Statistical AnalysisAll experiments were repeated at least 3 times. Data are presented as mean±SEM. Data analysis involved one-way ANOVA with LSD post-hoc test. P<0.05 was considered statistically significant.ResultsOx-LDL upregulates the expression of DKK1 in macrophagesCompared with the control groups, stimulation with 100 mg/L ox-LDL significantly increased both the protein and mRNA expression of DKK1 at 6 hr (P<0.05) and significantly increased the secretion of DKK1 in culture medium at 9 hr (P<0.05).The canonical Wnt pathway involves in the regulation of DKK1 by ox-LDLWe blocked the expression of β-catenin in macrophages by siRNA and detected the expression of CyclinD1 as its target gene (P<0.05). Compared with the negative control groups, β-catenin siRNA knockdown reduced the ox-LDL-increased protein level of DKK1 in macrophages (P<0.05).LXRα rather than LXRβ involves in the regulation of DKK1 by ox-LDLPretreatment with the LXRs agonists T0901317, GW3965 and 22-(R)-hydroxycholesterol abolished the ox-LDL-increased DKK1 expression (P<0.05). The LXRs antagonists GGPP and 22-(S)-hydroxycholesterol increased the expression of DKK1 (P<0.05), which resembled the upregulation of DKK1 by ox-LDL. Knocking down LXRa level by siRNA increased the level of DKK1 as well (P<0.05). However, the function of LXRβ had no significant effect on regulation of DKK1 by ox-LDL (P>0.05). Ox-LDL inhibits the interaction of LXRa and β-catenin in macrophagesImmunoprecipitation assay revealed that ox-LDL stimulation not only decreased the interaction of LXRa and β-catenin, but also inhibited the interaction of LXRβ and β-catenin.ConclusionsOx-LDL increases the expression of DKK1 in macrophages. Ox-LDL increases DKK1 by decreasing the repression of LXRa on β-catenin.BackgroundWnt signaling pathway was first identified for its role in carcinogenesis, but has since been recognized for its function in embryonic development. Recently, the functions of Wnt signaling pathway on cardiovascular diseases were paid more and more attention. DKK1, a secreted inhibitor of the canonical Wnt pathway, involves in the regulation of skin pigmentation and thickness, bone formation and homeostasis, neurodegenerative disorders, adipogenesis. The influence of DKK1 on cardiovascular diseases can’t be ignored. DKK1 affects the formation of kinds of cardiovascular tissues such as myocardial cell and heart valves. DKK1 also promote the endothelial-mesenchymal transition in aortic endothelial cells, which regulates the development of atherosclerosis. At the same time, DKK1 increases the inflammatory reaction between endothelium and platelet, promoting the incidence of atherosclerosis and plaque rupture.DKK1 is closely related with lipid metabolism. It is reported that DKK1 can promote adipogenesis. The impaired adipogenesis in hypertrophic obesity is mainly due to an inability to suppress canonical WNT pathway and to induce DKK1. While the relationship between DKK1 and lipid metabolism of macrophages in atherosclerosis still be unknown.Macrophages transform into foam cells with phagocytizing lipid, which play an important role in atherosclerosis. The transport of lipid in macrophages consists of three procedures, lipid phagocytic, transformation and reverse cholesterol transport. Many lipid receptors control this process. Lipoproteins and lipoprotein particles were recognized by lipid receptors on the membrane and englobed into the cells. Then lipids were transported to lysosomes and broken into amino acids and free cholesterol. Acyl coenzyme A cholesterol acyltransferase (ACAT) transformed them into cholesteryl ester for storage. When there were HDL and apoA-I, intracellular free cholesterol was reverse transported by ABCA1 and ABCG1 to avoid excessive lipid deposition.ObjectivesWe investigated whether DKK1 influences lipid metabolism of macrophages and the role of LOX-1 and ABCA/G1 in the regulation and the underlying mechanism.Materials and MethodsMaterialsRecombinant human DKK1 (rDKKl) was from R&D Systems (Minneapolis, MN, USA). Antibodies for phosophorylated-β-catenin (p-β-catenin),β-catenin, p-signal transducer and activator of transcription 3 (p-STAT3), STAT3, P-actin were from Cell Signaling Technology (Danvers, MA, USA). Antibody for ABCG1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for ABCA1 and DKK1 were from Abcam (Cambridge, UK). Antibody for LOX-1 was from Epitomics (Hangzhou, China). Stattic was from Selleckchem (Shanghai). Dil-labeled ox-LDL (Dil-ox-LDL) and ox-LDL were from Yiyuan Biotechnologies (Guangzhou, China).Cell CultureTHP-1 cells were grown in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO2-humidified incubator at 37℃ For differentiation into macrophages, THP-1 cells were seeded in culture plates and allowed to adhere and differentiate overnight in the presence of 160 nM PMA.RNA interferenceMacrophages cultured with antibiotic-free medium were transfected with specific siRNA or negative control siRNA by using Lipo-2000. After 6 hr, complete culture medium was replaced. Gene silencing efficiency was determined by western blot analysis.Western blotCells were lysed with RIPA lysis buffer including 1 mM PMSF. Extracted proteins were separated on SDS-PAGE gel and transferred to PVDF membranes for incubation with primary antibodies overnight at 4℃. The bands were visualized by enhanced chemiluminescence reagents and recorded by use of the LAS-4000 luminescent image analyzer.Foam cell formation and Dil-ox-LDL uptake assayDifferentiated THP-1 cells were cultured on slides and loaded with ox-LDL. After siRNA transfection or rDKKl incubation, cells were fixed with 4% paraformaldehyde and stained with Oil Red O and counterstained with hematoxylin. Images were obtained by light microscopy to assess foam cell formation.Dil-ox-LDL, labeled with red fluorescence, has been used to measure ox-LDL uptake by macrophages. Differentiated THP-1 cells were cultured on Lab-Tek Ⅱ chamber slides and loaded with Dil-ox-LDL. After siRNA transfection or rDKK1 incubation, cells were fixed with 4% paraformaldehyde and counterstained with DAPI. Cells were examined by confocal microscopy at 549 nm excitation and 565 nm emission.Statistical AnalysisAll experiments were repeated at least 3 times. Data are presented as mean±SEM. Data analysis involved unpaired t-test and one-way ANOVA with LSD post-hoc test. P<0.05 was considered statistically significant.ResultsDKK1 inhibits lipid accumulation and foam cell formation in macrophagesIntracellular lipid droplet accumulation was greater with DKK1 siRNA knockdown than the negative control (P<0.05). To confirm our findings, confocal microscopy revealed that DKKl siRNA knockdown significantly increased intracellular Dil-ox-LDL level (P<0.05).Pretreated cells with 100 ng/mL rDKKl and 100 mg/L ox-LDL, intracellular lipid droplet accumulation was decreased (P<0.05) as was intracellular Dil-ox-LDL level (P<0.05),Effect of DKKl on Wnt/p-catenin pathway in macrophagesDKK1 siRNA knockdown increased the expression of p-catenin and decreased its phosphorylation in macrophages (P<0.05), which was in contrast with the effects of rDKK1.Intracellular lipid droplet accumulation and Dil-ox-LDL level were both decreased with P-catenin siRNA knockdown (P<0.05), so DKKl suppressed lipid accumulation in part via a Wnt/β-catenin pathway.DKKl inhibits the expression of LOX-1 via Wnt/p-catenin pathwayWe found that rDKK1 abolished the ox-LDL-increased expression of LOX-1 (P<0.05) and DKK1 siRNA knockdown increased the expression (P<0.05).P-catenin siRNA knockdown blocked the ox-LDL-increased expression of LOX-1, which resembled the rDKK1 effect (P<0.05).DKKl increases the expression of ABCA/G1 via STAT3 pathwayPhosphorylation of STAT3 was increased in macrophages incubated with rDKK1 since 10 min (P<0.05). We used Stattic, a selective STAT3 inhibitor, to inhibit STAT3 phosphorylation (P<0.05), and found a significantly impaired effect of rDKKl on ABCA/G1 expression (P<0.05). SiRNA knockdown of STAT3 expression resulted in abolished upregulation of ABCA1 expression by rDKK1 (P<0.05).ConclusionsDKK1 inhibits intracellular lipid accumulation in macrophages. DKK1 affects the expression of LOX-1 and ABCA/G1 via a Wnt and STAT3 pathway, respectively.BackgroundAutophagy, a conservative phenomenon during eukaryotic evolution, means the process of hydrolysis in lysosomal for intracellular content. Autophagy consists of macroautophayg, microautophagy and chaperone-mediated autophagy. Macroautophagy is triggered by starvation, inflammation and hypoxia, and the double membranes of phagosomes obtain cellular debris to digest for recycling. Microautophagy means that the intracellular debris were gotten by lysomomal and degraded. Chaperone-mediated autophagy needs the assistance of molecule chaperone.Autophagy has a dual role in tumorigenesis. In the early stage of tumorigenesis, autophagy can block the development of tumor, and inhibiting autophagy promotes the tumor growth. On the other hand, along with the rapid growth of tumor, there are ischemia and hypoxia, which induces autophagy and causes apoptosis and necrosis of tumor cells. Hence, autophagy promotes the development of tumors.The essence of autophagy is a membrane transport system, through the formation of the double membranes structure. It is reported that phagosomes can combine with lipid droplet to form lipophagy. Lipophagy will move to lysomomal and fuse it. Then cholesteryl ester droplets are degraded into free cholesterol and transported outside.Researchers found that Wnt/β-catenin pathway plays a negative regulatory role for autophagy in basic and stress state. When autophagy is activated, β-catenin will be autophagic degraded and inhibits Wnt pathway in turn. The relationship between autophagy and Wnt/β-catenin pathway forms a negative feedback loop. Whether autophagy involved in the regulation of lipid metabolism by DKK1 has not been reported.ObjectivesWe investigated whether DKK1 influences autophagy and the role of autophagy in the regulation of lipid metabolism by DKK1.Materials and MethodsMaterialsRecombinant human DKK1 (rDKK1) was from R&D Systems (Minneapolis, MN, USA). Antibodies for LC3B, ATG5, p-catenin, β-actin and LY294002, MG-132 were from Cell Signaling Technology (Danvers, MA, USA). Antibody for ABCG1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody for ABCA1 was from Abcam (Cambridge, UK). Antibody for LOX-1 was from Epitomics (Hangzhou, China). Rapamypin was from Selleckchem (Shanghai). Dil-labeled ac-LDL (Dil-ac-LDL), ac-LDL and ox-LDL were from Yiyuan Biotechnologies (Guangzhou, China).Cell CultureTHP-1 cells were grown in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. For differentiation into macrophages, THP-1 cells were allowed to adhere and differentiate with 160 nM PMA.RNA interferenceMacrophages cultured with antibiotic-free medium were transfected with specific siRNA or negative control siRNA by using Lipo-2000. After 6 hr, complete culture medium was replaced. Gene silencing efficiency was determined by western blot.Western blotCells were lysed with RIPA lysis buffer including 1 mM PMSF.Extracted proteins were separated on SDS-PAGE gel and transferred to PVDF membranes for incubation with primary antibodies overnight at 4℃.The bands were visualized by enhanced chemiluminescence reagents and recorded by use of the LAS-4000 luminescent image analyzer.Foam cell formation and Dil-ox-LDL uptake assayDifferentiated THP-1 cells were cultured on slides and loaded with ac-LDL. After siRNA transfection or rDKK1 incubation, cells were fixed with 4% paraformaldehyde and stained with Oil Red O and counterstained with hematoxylin. Images were obtained by light microscopy to assess foam cell formation.Dil-ac-LDL, labeled with red fluorescence, has been used to measure ac-LDL uptake by macrophages. Differentiated THP-1 cells were cultured on Lab-Tek II chamber slides and loaded with Dil-ac-LDL. After siRNA transfection or rDKKl incubation, cells were fixed with 4% parafonnaldehyde and counterstained with DAPI. Cells were examined by confocal microscopy at 549 nm excitation and 565 nm emission.ImmunoprecipitationDifferentiated THP-1 cells were incubated with or without rDKKl, and lysed with lysis buffer for western blot analysis and immunoprecipitation. Cell lysates were incubated with cognate antibodies and 50% protein A/G agarose overnight at 4℃ with gentle rotation. Agarose-bound immunoprecipitates were rinsed and underwent SDS-PAGE (2×) before western blot analysis.Statistical AnalysisAll experiments were repeated at least 3 times. Data are presented as mean±SEM. Data analysis involved one-way ANOVA with LSD post-hoc test. P<0.05 was considered statistically significant.ResultsDKK1 increases the level of autophagy in macrophagesThe level of autophagy is low at basic state, and autophagy inducers ox-LDL, Rapamycin, EBSS, lead to significantly increase of LC3B-II and the level of autophagy (P<0.05). Then with addition of rDKK1, the level of LC3B-Ⅱ and autophagy is much higher (P<0.05). Blockage of DKK1 by DKK1 siRNA, compared with negative control groups, the expression of LC3B-II is significantly decreased (P<0.05). Autophagy inhibitors LY294002 and 3-MA abolish rDKKl-induced autophagy (P<0.05).Autophagy involved in the regulation of lipid metabolism by DKK1When autophagy is inhibited by inhibitor 3-MA or ATG5 siRNA, rDKKl can’t inhibit lipid accumulation in macrophages (P<.05). Confocal microscopy reveals that rDKK1 doesn’t decrease intracellular Dil-ox-LDL level with autophagy blockage (P<0.05).Autophagy affects the expression of lipid receptors by DKK1Blockage of autophagy by inhibitor 3-MA or ATG5 siRNA, rDKK1 can’t decrease the expression of LOX-1 (P<0.05). The increase of ABCA/G1 by DKK1 was abolished when autophagy is down regulated (P<0.05).DKK1 inhibits the expression of β-catenin by autophagic degradationAutophagy inhibitor 3-MA can block the decrease of β-catenin by DKK1, resembling the proteasome inhibitor MG-132 (P<0.05). Compared with negative control group, knockdown ATG5 by siRNA has a significantly impaired effect of DKK1 on β-catenin (P<0.05). Immunoprecipitation assay reveals that rDKK1 stimulation increases the interaction of LC3 and β-catenin, which may result in the autophagic degradation of P-catenin.ConclusionsDKK1 increases autophagy and autophagic degradation of P-catenin. Autophagy involved in the regulation of lipid metabolism and lipid receptors by DKK1 in macrophages.